The invention relates to a new class of epothilone derivatives, the production of these compounds and new intermediates, pharmaceutical preparations containing these compounds, and the use of these compounds in the treatment of warm-blooded animals, such as humans, or their use in the production of pharmaceutical preparations for the treatment of warm-blooded animals, such as humans.
Epothilones A and B represent a new class of microtubule-stabilising cytotoxic agents (see Gerth, K. et al., J. Antibiot. 49, 560-3 (1966)) of the formulae: 
wherein R means hydrogen (epothilone A) or methyl (epothilone B).
These compounds have advantages over Taxol(copyright), a branded product already introduced for the treatment of tumours, that has the same mechanism of action but has however a series of disadvantages, such as very poor water solubility, making the preparation of pharmaceutical formulations very difficult (at present, such formulations are normally characterised by the toxic side effects of the carrier materials), and inefficacy on a series of tumours. The advantages are as follows:
a) They have better water-solubility and are thus more readily accessible for formulations.
b) It has been reported that, in cell culture experiments, they are also active against the proliferation of cells, which, owing to the activity of the P-glycoprotein efflux pump making them xe2x80x9cmultidrug resistantxe2x80x9d, show resistance to treatment with other chemotherapy agents including Taxol(copyright) (see Bolag, D. M., et al., xe2x80x9cEpothilones, a new class of microtubule-stabilizing agents with a Taxol-like mechanism of actionxe2x80x9d, Cancer Research 55, 2325-33 (1995)). And
c) it could be shown that they are still very effective in vitro against a Taxol(copyright)-resistant ovarian carcinoma cell line with modified xcex2-tubulin (see siehe Kowalski, R. J., et al., J. Biol. Chem. 272(4), 2534-2541 (1997)).
Pharmaceutical application of the epothilones, for example for tumour treatment, is possible in an analogous manner to that described for Taxol, see for example U.S. Pat. No. 5.641.803; U.S. Pat. No. 5.496.804; U.S. Pat. No. 5.565.478). One disadvantage of the epothilones is the relatively low therapeutic index, i.e. the dosage range between the necessary dose and the maximum tolerable dose is very small.
In the meantime, a series of epothilone derivatives have been published in the search for new, more effective and more versatile products.
Up until now, all epothilones described in the literature contain a methyl group shown in the above formula at C-16. This methyl group (C-17) was hypothesized to be necessary in order Ws to force the heterocycle (in the case of epothilones A and B a methylthiazolyl ring) out of the plane of the conjugated double bond between C-16 and C-18, and this was postulated to be necessary for efficacy.
It is an objective of the invention to provide a new class of epothilone derivatives, which have a new type of structure and which, through their advantageous biological and pharmacological properties, enable the armamentarium for the control of, in particular, proliferative diseases such as tumours to be expanded. Also, compounds must be found which have an improved therapeutic index compared with epothilones A and B.
Surprisingly, a new class of epothilone derivatives has been found, which are pharmacologically highly effective, despite the absence of the methyl group on C-16 of the epothilones and even though the resulting heterocyclic ring lies on the plane of the C-16/C-18 double bond.
The invention relates to this new class of epothilone derivatives. The compounds in question are compounds of formula I, 
wherein
T is O, NH or N(alkyl), wherein alkyl is alkyl, especially lower alkyl;
as A is a radical of formula Ia 
xe2x80x83which is bonded to the radical of the molecule according to formula I by one of the two carbon atoms marked with an arrow, especially by the lower carbon atom (the one nearer to the nitrogen in the fused heterocycylic ring or para to the atom X), and wherein
X is S; O; NH; N(alk); wherein alk is alkyl, hydroxy-lower alkyl, unsubstituted or substituted amino-lower alkyl or carbamoyl-lower alkyl; N(ar), wherein ar is aryl; C(Rk*)xe2x95x90N, Nxe2x95x90C(Rk*) or C(Rk*)xe2x95x90C(Rk**), wherein Rk* and Rk**, independently of one another, are H, alkyl (especially lower alkyl), unsubstituted or substituted amino-lower alkyl, carbamoyl-lower alkyl, or in particular halogen-lower alkyl or hydroxy-lower alkyl, or further aminolower alkyl; and
Rk is H, alkyl (especially lower alkyl), unsubstituted or substituted amino-lower alkyl, carbamoyl-lower alkyl, or in particular halogen-lower alkyl or hydroxy-lower alkyl;
either Y is OH and Y* is hydrogen, or xe2x80x94Y and xe2x80x94Y* together form a bond (so that together with the adjoining bond connecting the two xe2x80x94Y and xe2x80x94Y* bearing carbon atoms they form a double bond);
R is hydrogen, lower alkyl or halogen-lower alkyl;
and Z is O, or xe2x80x94Zxe2x80x94 is a bond between the two binding carbon atoms;
or salts thereof.
These compounds have advantageous pharmaceutical properties. For example, they are active against multidrug-resistant cell lines and tumours and/or they have an improved therapeutic index over natural epothilones.
The general terms used hereinbefore and hereinafter preferably have within the context of this disclosure the following meanings, unless otherwise indicated:
The prefix xe2x80x9clowerxe2x80x9d denotes a radical having up to and including a maximum of 7, especially up to and including a maximum of 4 carbon atoms, the radicals in question being either unbranched or branched with single or multiple branching.
Where the plural form is used for compounds, salts, and the like, this is taken to mean also a single compound, salt, or the like (xe2x80x9caxe2x80x9d as an indefinite article or as a numeral meaning xe2x80x9conexe2x80x9d).
Asymmetric carbon atoms that are optionally present in the substituents may exist in the (R), (S) or (R,S) configuration, preferably in the (R) or (S) configuration. Substituents on a double bond or on a ring, for example on the carbon atoms to which Z in formula I is bonded, may be present in cis- (=Z-) or trans- (=E-) form. The present compounds may thus exist as mixtures of isomers or as pure isomers, preferably as pure diastereoisomers.
T is either O, NH or N(alkyl), especially either O or NH, preferably O.
The radical A is bonded to the radical of the molecule of formula I by one of the two carbon atoms marked by an arrow in formula Ia. Bonding is preferably effected via the carbon atom of the benzene ring portion of A which is in para position to X.
Alkyl is preferably an alkyl radical with 1 to 10 carbon atoms, preferably lower alkyl, especially methyl.
Lower alkyl is unbranched or has mono- or multiple-branching and is in particular methyl or ethyl.
Aryl is preferably an aromatic radical with 6 to 14 carbon atoms, especially phenyl, naphthyl, fluorenyl or phenanthrenyl, whereby the said radical is unsubstituted or is substituted by one or more substituents, preferably up to three, primarily one or two substituents, especially those selected from amino; lower alkanoylamino, especially acetylamino; halogen, especially fluorine, chlorine or bromine; lower alkyl, especially methyl or also ethyl or propyl; halogen-lower alkyl, especially trifluoromethyl; hydroxy; lower alkoxy, especially methoxy or also ethoxy; phenyl-lower alkoxy, especially benzyloxy; nitro, cyano, C8-C12-alkoxy, especially n-decyloxy, carbamoyl, lower alkyl-carbamoyl, such as N-methyl- or N-tert-butylcarbamoyl, lower alkanoyl, such as acetyl, phenyloxy, halogen-lower alkyloxy, such as trifluoromethoxy or 1,1,2,2-tetrafluoroethyloxy, lower alkoxycarbonyl, such as ethoxycarbonyl, lower alkylmercapto, such as methylmercapto, halogen-lower alkyl-mercapto, such as trifluoromethylmercapto, hydroxy-lower alkyl, such as hydroxymethyl or 1-hydroxymethyl, lower alkanesulphonyl, such as methanesulphonyl, halogen-lower alkane-sulphonyl, such as trifluoromethanesulphonyl, phenylsulphonyl, dihydroxybora (-B(OH)2), 2-methyl-pyrimidin-4-yl, oxazol-5-yl, 2-methyl-1,3-dioxolan-2-yl, 1H-pyrazol-3-yl, 1-methyl-pyrazol-3-yl; and lower alkylenedioxy which is bonded to two adjacent carbon atoms, such as methylenedioxy.
Halogen is especially fluorine, chlorine, bromine, or iodine, in particular fluorine or chlorine.
Halogen lower alkyl is methyl or ethyl that is substituted in particular by halogen, such as fluorine or chlorine, especially fluoromethyl or also chloromethyl.
Hydroxy lower alkyl is in particular lower alkyl that is terminally substituted by hydroxy, preferably hydroxymethyl or hydroxyethyl, especially 2-hydroxyethyl.
Unsubstituted or substituted amino lower alkyl is in particular lower alkyl that is terminally substituted by amino or substituted amino, whereby preferably one or two lower alkyl radicals are present as amino substituents; aminomethyl, 2-aminoethyl, N-methylamino-methyl, 2-(N-methylamino)ethyl, N,N-dimethylaminomethyl or 2-(N,N-dimethylamino)ethyl are preferred.
Carbamoyl lower alkyl is in particular lower alkyl that is terminally substituted by xe2x80x94C(O)NH2, preferably carbamoylmethyl or 2-carbamoylethyl.
Where these radicals are present, one of radicals Rk, Rk* and Rk** is preferably hydrogen and the others are also each hydrogen, or preferably one is lower alkyl, such as methyl, halogen-lower alkyl, such as methyl fluoride, or hydroxy-lower alkyl, such as hydroxymethyl or -ethyl, and the other is hydrogen, or one is unsubstituted or substituted amino lower alkyl, especially aminoethyl, 2-aminoethyl, N-methylaminomethyl, 2-(N-methylamino)ethyl, N,N-dimethylaminomethyl or 2-(N,N-dimethylamino)ethyl, and the other is hydrogen,
xe2x80x94Y and xe2x80x94Y* either form a bond, so that a double bond exists together with the bond linking the Y and Y* bearing carbon atoms; or preferably Y is hydroxy and Y* is hydrogen.
Where present, one of radicals Rk, Rk* and Rk** is preferably methyl or hydroxymethyl, and the other(s) is or are hydrogen, or one of radicals Rk, Rk* and Rk** is 2-hydroxyethyl, aminomethyl, 2-aminoethyl, N,N-dimethylaminomethyl or carbamoylmethyl, and the other(s) is or are hydrogen.
If Rk* and Rk** are not present, Rk is preferably hydrogen, methyl or hydroxymethyl, or ethyl.
R is preferably hydrogen, methyl or fluoromethyl, also ethyl.
Z is either O, whereupon an oxirane is formed with the binding carbon atoms, or xe2x80x94Zxe2x80x94 is a bond, so that a double bond is produced together with the already existing bond between the two carbon atoms to which Z is bonded.
The wavy line of the bond to the Z-bearing carbon atom is to indicate that the compound of formula I, in respect of the epoxide ring or the double bond formed by Z and the two adjacent carbon atoms, may be present in the Z- (=cis-)form or in the E- (=trans-)form, furthermore as a mixture of these forms, the Z-form being preferred.
Salts are primarily the pharmaceutically acceptable salts of compounds of formula I.
Such salts are formed, for example, as acid addition salts, preferably with organic or inorganic acids, from compounds of formula I with a basic nitrogen atom, especially the pharmaceutically acceptable salts. Suitable inorganic acids are, for example, hydrohalic acids, such as hydrochloric acid, sulphuric acid, or phosphoric acid. Suitable organic acids are, for example, carboxylic, phosphonic, sulphonic or sulphamic acids, for example acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, 2-hydroxybutyric acid, gluconic acid, glucosemonocarboxylic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, malic acid, tartaric acid, citric acid, glucaric acid, galactaric acid, amino acids, such as glutamic acid, aspartic acid, N-methylglycine, acetylaminoacetic acid, N-acetylasparagine or N-acetylcysteine, pyruvic acid, acetoacetic acid, phosphoserine, 2- or 3-glycerophosphoric acid, maleic acid, hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid, benzoic acid, salicylic acid, 1- or 3-hydroxy-naphthyl-2-carboxylic acid, 3,4,5-trimethoxybenzoic acid, 2-phenoxybenzoic acid, 2-aceto-xybenzoic acid, 4-aminosalicylic acid, phthalic acid, phenylacetic acid, glucuronic acid, galacturonic acid, methane- or ethane-sulphonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulphonic acid, benzenesulphonic acid, 2-naphthalenesulphonic acid, 1,5-naphthalene-disulphonic acid, N-cyclohexylsulphamic acid, N-methyl-, N-ethyl- or N-propyl-sulphamic acid, or other organic protonic acids, such as ascorbic acid.
For isolation or purification purposes it is also possible to use pharmaceutically unacceptable salts, for example picrates or perchlorates. Only the pharmaceutically acceptable salts or free compounds (if the occasion arises, in the form of pharmaceutical preparations) attain therapeutic use, and these are therefore preferred.
In view of the close relationship between the novel compounds in free form and in the form of their salts, including those salts that can be used as intermediates, for example In the purification or identification of the novel compounds, hereinbefore and hereinafter any reference to the free compounds is to be understood as referring also to the corresponding salts, as appropriate and expedient.
The compounds of formula I have valuable pharmacological properties, as described hereinbefore and hereinafter.
The efficacy of the compounds of formula I as inhibitors of microtubule depolymerisation may be proved as follows:
Stock solutions of the test compounds (10 mM) are prepared in DMSO and stored at xe2x88x9220xc2x0 C. Microtubule protein is extracted from pigs"" brain by two cycles of temperature-dependent depolymerisation/polymerisation, as known (see siehe Weingarten et al., Biochemistry 1974; 13: 5529-37). Working stock solutions of microtubule protein (i.e. tubulin plus microtubule-associated proteins) are stored at xe2x88x9270xc2x0 C. The degree of test-compound-induced polymerisation of microtubule protein is determined basically as already known (see Lin et al., Cancer Chem. Pharm. 1996; 38:136-140). To summarise, 5 xcexcl stock solution of the test compound in 20 times the desired final concentration are admixed with 45 xcexcl of water at room temperature and then placed on ice. A working aliquot strain of pigs"" brain microtubule protein is rapidly thawed and then diluted to 2 mg/ml in ice-cold 2xc3x97MEM buffer (200 ml MES, 2 mM EGTA, 2 mM MgCl2, pH 6.7) [MES=2-morpholinoethanesulphonic acid, EGTA=ethylene glycol-bis-2(2-aminoethyl)-tetraacetic acid]. The polymerisation reaction is started by adding 50 xcexcl if of each diluted microtubule protein to the test compound, followed by incubation of the sample for 5 minutes in a water bath at room temperature. Then, the reaction mixtures are placed in an Eppendorf microcentrifuge and incubated for a further 15 minutes at room temperature. The samples are then centrifuged for 20 minutes at 14,000 rpm at room temperature, in order to separate polymerised from unpolymerised microtubule protein. As an indirect measure of tubulin polymerisation, the protein concentration of the supernatant (which contains the remaining non-polymerised, soluble microtubule protein) is determined by the Lowry method (CD Assay Kit, Bio-Rad Laboratories, Hercules, Calif.), and the optical density (OD) of the colour reaction is measured at 750 nm using a spectrometer (SpectraMax 340, Molecular Devices, Sunnydale, Calif.). The difference in OD""s between samples treated with a test compound and vehicle-treated controls is compared with that obtained with incubations containing 25 xcexcM epothilon B (positive control). The degree of polymerisation induced by a test compound is expressed relatively to the positive control (100%). By comparing the activity of several concentrations, the EC50 (concentration at which 50% of the maximum polymerisation occurs) can be determined. For compounds of formula I, the EC50 lies in the range of 1 to 100, preferably in the range of 1 to 50, especially from 1 to 10 xcexcM.
The efficacy against tumour cells may be demonstrated in the following way:
Stock solutions of the test compounds (10 mM) are prepared in DMSO and stored at xe2x88x9220xc2x0 C. Human KB-31 and (multidrug-resistant, P-gp170 expressing) KB-8511 epidermoid carcinoma cells originate from Dr. M. Baker, Roswell Park Memorial Institute (Buffalo, N.Y., USA) (description: see also Akiyama et al., Somat. Cell. Mol. Genetics 11 117-120 (1985) and Fojo A., et al., Cancer Res. 45, 3002-3007 (1985)xe2x80x94KB-31 and KB-8511 are both derivatives of the KB cell line (ATCC) and are human epidermis carcinoma cells. KB 31 cells may be cultivated in mono-layers using Dulbecco""s modified Eagle""s medium (D-MEM) with 10% foetal calf serum (M.A. Bioproducts), L-glutamine (Flow), penicillin (50 units/ml) and streptomycin (50 xcexcg/ml (Flow); they then grow at a duplication time of ca. 22 hours, and their relative plating efficiency it ca. 60%. KB-8511 is a variant derived from the KB-31 cell line, which was obtained using colchichine treatment cycles, and has an approximately 40 times relative resistance to colchichine compared with KB-31 cells. The cells are incubated at 37xc2x0 C. in an incubator with 5% v/v CO2 and at 80% relative humidity with MEMAlpha medium which contains ribonucleosides and desoxyribonucleosides (Gibco BRL), complemented with 10 IU penicillin, 10 xcexcg/ml streptomycin and 5% foetal calf serum. The cells are seeded in a quantity of 1.5xc3x97103 cells/well in 96-well microtitre plates, and incubated over night. Serial dilutions of the test compounds in culture medium are added on day 1. The plates are then incubated for a further 4 days, after which the cells are fixed with 3.3% v/v glutaraldehyde, washed with water and dyed with 0.05% w/v methylene blue. After washing, the dye is eluted with 3% HCl and the optical density measured at 665 nm with a SpectraMax 340 (Molecular Devices, Sunnyvale, Calif.). IC50 values are determined by adaptation of mathematical curves, using the SofrPro2.0 programme (Molecular Devices, Sunnyvale, Calif.) and using the formula [(OD treated)xe2x80x94(OD start) [/](OD control)xe2x80x94(OD start)]xc3x97100. The IC50 is defined as the concentration of a test compound at the end of the incubation period, which led to 50% of the cell count per well compared with the control (concentration at semi-maximum inhibition of cell growth). Compounds of formula I thus preferably show an IC50 in the range of 0.1xc3x9710xe2x88x929 to 500xc3x9710xe2x88x929M, preferably between 0.2 and 50 nM.
Tests on other tumour cells lines can also be carried out in a comparable manner. The ranges of the IC50 values (the ranges measured for compounds of the formula I, especially for the preferred compounds of formula I) are given in the square parentheses. A459 (lungs; ATCC CCL 185) [preferred IC50 0.01xc3x9710xe2x88x929 to 500xc3x9710xe2x88x929M, preferably between 0.01 and 100 nM], NClH460 (lungs) [preferred IC50 0.01xc3x9710xe2x88x929 to 500xc3x9710xe2x88x929M, preferably between 0.02 and 200 nM], HCT-15 (colon; ATCC CCL 225xe2x80x94ATCC=American Type Culture Collection (Rockville, Mass., USA)) [preferred IC50 0.01xc3x9710xe2x88x929 to 500xc3x9710xe2x88x929M, preferably between 0.05 and 500 nM], HCT-116 (colon) [preferred IC50 0.01xc3x9710xe2x88x929 to 500xc3x9710xe2x88x929M, preferably between 0.05 and 200 nM], Du145 (prostate; ATCC No. HTB 81; see also Cancer Res. 37, 4049-58 [1978]) [preferred IC50 0.01xc3x9710xe2x88x929 to 500xc3x9710xe2x88x929M, preferably between 0.05 and 500 nM], PC-3M (prostatexe2x80x94hormone-insensitive derivative, obtained from Dr. I. J. Fidler (MD Anderson Cancer Center, Houston, Tex., USA) and derived from PC-3, a cell line that is obtainable from the ATCC (ATCC,CRL 1435)) [preferred IC50 0.01xc3x9710xe2x88x929 to 500xc3x9710xe2x88x929M, preferably between 0.05 and 500 nM], MCF-7 (breast; ATCC HTB 22) [preferred IC50 0.01xc3x9710xe2x88x929 to 500xc3x9710xe2x88x929M, preferably between 0.02 and 200 nM], MCF-7/ADR (breast, multidrug-resistant; see also Blobe G.C.et al., J. Biol. Chem. (1983), 658-664; the cell line is to a large extent resistant (360- to 2400-fold) to doxorubicin and vinca alkaloids in comparison with MDR-7 xe2x80x9cwild-typexe2x80x9d cells)) [preferred IC50 0.01xc3x9710xe2x88x929 to 500xc3x9710xe2x88x929M, preferably between 0.1 and 1000 nM], or MDA231.
The in vivo efficacy may be demonstrated as follows: The models used are xeno-transplants of tumours, such as KB-31 or KB-8511 epidermoid tumours, in mice. The anti-tumour efficacy of the test compounds may be measured in female BLB/c nu/nu mice for example against the corresponding subcutaneously transplanted cell line. To this end, tumour fragments of about 25 mg are implanted into the left side of each of the mice (for example 6 animals per dose). The test compound is administered for example on day 11 after transplantation in different dosages (for example 0.1; 0.5; 1; 5 and 10 mg/kg), if desired repeating the administration, if required several times, after between two days and two weeks. The volumes of the tumours are determined for example after about 2 to 4 weeks (e.g. two weeks after the start of treatment). The tumour volumes are calculated by measuring the tumour diameter along two vertically arranged axes and according to published methods (see Evans et al., Brit. J. Cancer 45, 466-8 (1982)). The anti-tumour efficacy is determined as the mean increase in tumour volume of the treated animals divided by the mean Increase in tumour volume of the untreated animals (controls) and, after multiplication by 100, is expressed as T/C %. Tumour regression (given in %) is calculated as the smallest mean tumour volume (Vt) in relation to the mean tumour volume at the start of treatment (Vo) according to the formula
% regression=[1xe2x88x92(Vt/Vo)]xc3x97100.
In this case also, other cell lines can be used, for example those named above in the demonstration of efficacy against tumour cells.
Owing to these properties, the compounds are suitable for the treatment of proliferative diseases, especially tumour diseases, including metastases; for example solid tumours such as lung tumours, breast tumours, colorectal tumours, prostate tumours, melanomas, brain tumours, pancreas tumours, neck tumours, bladder tumours, neuroblastomas, throat tumours, but also proliferative diseases of blood cells, such as leukaemia; also for the treatment of other diseases which respond to treatment with microtubule depolymerisation inhibitors, such as psoriasis.
A compound of formula I can be administered alone or in combination with one or more other therapeutic agents; possible combination therapy taking the form of fixed combinations or the administration of a compound of the invention and one or more other therapeutic agents being staggered or given independently of one another, or the combined administration of fixed combinations and one or more other therapeutic agents. A compound of formula I can besides or in addition be administered for tumour therapy in combination with chemotherapy, radiotherapy, immunotherapy, surgical intervention, or a combination of these. Long-term therapy is equally possible as is adjuvant therapy in the context of other treatment strategies, as described above. Other possible treatments are therapy to maintain the patient""s status after tumour regression, or even chemopreventive therapy, for example in patients at risk.
Therapeutic agents for possible combination are especially one or more antiproliferative, cytostatic or cytotoxic compounds, for example one or more chemotherapeutic agent(s) selected from the group comprising the classical chemotherapeutic agents, an inhibitor of polyamine biosynthesis, an inhibitor of protein kinase, especially of serine/threonine protein kinase, such as protein kinase C, or of tyrosine protein kinase, such as epidermal growth factor receptor protein tyrosine kinase, a cytokine, a negative growth regulator, such as TGF-xcex2 or IFN-xcex2, an aromatase inhibitor, and a classical cytostatic.
Compounds according to the invention are not only for the (prophylactic and preferably therapeutic) treatment of humans, but also for the treatment of other warm-blooded animals, for example of commercially useful animals, for example rodents, such as mice, rabbits or rats, or guinea-pigs. They may also be used as a reference standard in the test systems described above to permit a comparison with other compounds.
A compound of formula I may also be used for diagnostic purposes, for example with tumours that have been obtained from warm-blooded animal xe2x80x9chostsxe2x80x9d, especially humans, and implanted into mice to test them for decreases in growth after treatment with such a compound, in order to investigate their sensitivity to the said compound and thus to improve the detection and determination of possible therapeutic methods for neoplastic diseases in the original host.
Within the groups of preferred compounds of formula I mentioned hereinafter, definitions of substituents from the general definitions mentioned hereinbefore may reasonably be used, for example, to replace more general definitions with more specific definitions or especially with definitions characterized as being preferred; the definitions characterised as being preferred, or exemplary (xe2x80x9ce.g.xe2x80x9d, xe2x80x9csuch asxe2x80x9d, xe2x80x9cfor examplexe2x80x9d, are preferred.
Preference is given to a compound of formula I,
wherein
T is O or NH;
A is a radical of formula Ia, which is bonded to the radical of the molecule of formula I by one of the two carbon atoms marked by an arrow, preferably by the carbon atom in p-position to X; and wherein
X is S; O; NH; N(alk); wherein alk is alkyl, (especially lower alkyl), hydroxy-lower alkyl, unsubstituted or substituted amino-lower alkyl or carbamoyl-lower alkyl; C(Rk*)xe2x95x90N, Nxe2x95x90C(Rk*) or C(Rk*)xe2x95x90C(Rk**), wherein Rk* and Rk**, independently of one another, are H, Alkyl (especially lower alkyl), halogen-lower alkyl or hydroxy-lower alkyl, or also substituted or unsubstituted amino-lower alkyl or carbamoyl-lower alkyl; and
Rk is H, lower alkyl, halogen-lower alkyl or hydroxy-lower alkyl or also unsubstituted or substituted amino-lower alkyl or carbamoyl-lower alkyl;
either Y is OH and Y* is hydrogen, or xe2x80x94Y and xe2x80x94Y* together form a bond;
R is hydrogen, lower alkyl or halogen lower alkyl;
and Z is O, or xe2x80x94Zxe2x80x94 is a bond between the two binding carbon atoms;
or salts thereof.
Preference is also given to a compound of formula I wherein
T is O;
A is a radical of formula Ia 
xe2x80x83which is bonded to the radical of the molecule according to formula I by one of the two carbon atoms marked with an arrow, and wherein
X is S; O; NH; N(alk); wherein alk is alkyl; N(ar), wherein ar is aryl; C(Rk*)xe2x95x90N, Nxe2x95x90C(Rk*) or C(Rk*)xe2x95x90C(Rk**), wherein Rk* and Rk**, independently of one another, are H, alkyl (especially lower alkyl), halogen-lower alkyl or hydroxy-lower alkyl; and Rk is H, alkyl (especially lower alkyl), halogen-lower alkyl or hydroxy-lower alkyl;
either Y is OH and Y* is hydrogen, or xe2x80x94Y and xe2x80x94Y* together form a bond (so that they form a double bond together with the adjoining bond connecting the two xe2x80x94Y and xe2x80x94Y* bearing carbon C atoms);
R is hydrogen, lower alkyl or halogen-lower alkyl;
and Z is O, or xe2x80x94Zxe2x80x94 is a bond between the two binding carbon atoms;
or salts thereof.
Special preference is given to a compound of formula I wherein
T is NH or especially O;
A is a radical of formula Ia, which is bonded to the radical of the molecule according to formula I by one of the two carbon atoms marked with an arrow, and wherein
X is S, O, NH, N(CH3), N(CH2CH2OH), N(CH2CH2NH2), N(CH2CH2N(CH3)2, N(CH2C(O)NH2), C(Rk*)xe2x95x90N or CHxe2x95x90C(Rk*), wherein Rk* is H, methyl, hydroxymethyl, (CH2CH2OH), (CH2CH2NH2), (CH2CH2N(CH3)2), (CH2C(O)NH2), or also fluoromethyl;
and
Rk is hydrogen, methyl, ethyl, hydroxymethyl, hydroxyethyl, aminomethyl, aminoethyl, dimethylaminomethyl, carbamoylmethyl or also fluoromethyl;
R is hydrogen, methyl, ethyl or fluoromethyl; and
Z is O, or xe2x80x94Zxe2x80x94 is a bond between the two binding carbon atoms;
or salts thereof; whereby the bond characterised by a wavy line means that the compound of formula I is present in cis- or trans-form, preferably in cis-form.
Special preference is given to a compound of formula I wherein
T is NH or especially O;
A is a radical of formula Ia, which is bonded to the radical of the molecule according to formula I by one of the two carbon atoms marked with an arrow, add wherein
X is S, O, NH, N(CH3) or N(CH2CH2OH), or isC(Rk*)xe2x95x90N or CHxe2x95x90C(Rk*), wherein Rk* is H, methyl or hydroxymethyl, or also fluoromethyl;
and
Rk is hydrogen, methyl or hydroxymethyl, or also fluoromethyl, or ethyl;
R is hydrogen, methyl, ethyl or fluoromethyl; and
Z is O, or xe2x80x94Zxe2x80x94 is a bond between the two binding carbon atoms;
or salts thereof; whereby the bond characterised by a wavy line means that the compound of formula I is present in cis- or transform, preferably in cis-form.
Particular preference is given to a compound of formula I wherein
T is NH or especially O;
A means a radical of formula Ia, which is bonded to the radical of the molecule according to formula I by one of the two carbon atoms marked with an arrow, and wherein
X is S; or is further selected from the group consisting of NCH3 and CHxe2x95x90CH and in a broader aspect of the invention O and N(CH2CH2OH);
and
Rk is H, methyl, ethyl, hydroxymethyl, hydroxyethyl, especially 2-hydroxyethyl, aminomethyl, aminoethyl, especially 2-aminoethyl, or carbamoyl, preferably methyl;
R is hydrogen, lower alkyl or halogen lower alkyl; and
Z is O, or xe2x80x94Zxe2x80x94 is a bond between the two binding carbon atoms, or a salt thereof.
Particular preference is given to a compound of formula I wherein
T is O;
A means a radical of formula Ia, which is bonded to the radical of the molecule according to formula I by one of the two carbon atoms marked with an arrow, and wherein
X is S; or is further selected from the group consisting of O, NCH3, CHxe2x95x90CH and N(CH2CH2OH);
and
Rk is H, methyl or hydroxymethyl, or ethyl, preferably methyl;
R is hydrogen, lower alkyl or halogen lower alkyl; and
Z is O, or xe2x80x94Zxe2x80x94 is a bond between the two binding carbon atoms, or a salt thereof.
Of the compounds and (where appropriate) groups including the compounds, as mentioned hereinbefore and hereinafter, the following are preferred in particular (the free compounds being understood to mean also the corresponding salts):
(a) compounds of formula I, wherein X is S;
(b) compounds of formula I, wherein X is O;
(c) compounds of formula I, wherein X is NH;
(d) compounds of formula I, wherein X is Nxe2x80x94CH3;
(e) compounds of formula I, wherein X is CHxe2x95x90C(Rk*), in which Rk* meansCH3 or CH2OH;
(f) compounds of formula I, wherein X is C(Rk**)xe2x95x90N, in which Rk** means CH3 or CH2OH;
(g) compounds of formula I, wherein Z is O [also provided that they fall within one of the definitions (a) to (f)];
(h) compounds of formula I, wherein xe2x80x94Zxe2x80x94 is a bond [also if they fall within one of the definitions (a) to (f)];
(i) compounds of formula I, wherein the bond characterised by a wavy line is present in such a way that the compound of formula I is in cis-form;
(j) compounds of formula I, wherein Y is OH and Y* is hydrogen; as well as, furthermore
(k) compounds of formula I, wherein X is N(CH2CH2OH);
(l) compounds of formula I, wherein X is N(CH2CH2NH2);
(m) compounds of formula I, wherein X is N(CH2C(O)NH2); further
(n) compounds of formula I, wherein X is C(Rk*)xe2x95x90CH, in which Rk* means CH3 or CH2OH;
(o) compounds of formula I, wherein T is O;
(p) compounds of formula I, wherein T is NH;
(q) compounds of formula I, wherein X is CHxe2x95x90C(Rk*), in which Rk*means H; or
(r) compounds of formula I, wherein X is C(Rk**)xe2x95x90N, in which Rk** means H.
In the compounds named under the above definitions (a) to (r), the remaining radicals respectively have the meanings given hereinbefore and hereinafter for compounds of formula I, especially those characterised as being preferred meanings. Of the compounds of formula I and their salts falling within definitions (a) to (r), particular preference is given to those in which the radical A of formula Ia is linked by the bond in para position to X (i.e.  is a radical of formula 
wherein the radicals X and Rk have the meanings given for compounds of formula I).
Very preferred is a compound of formula I, in which T is NH or especially O;  is a radical of formula 
wherein X is sulphur and Rk is methyl, Z is oxygen or xe2x80x94Zxe2x80x94 is a bond, and Y* and Y* or xe2x80x94Y and xe2x80x94Y* have the above meanings, especially either Y is hydroxy and Y* is hydrogen, or especially xe2x80x94Y and xe2x80x94Y* together form a bond, or a salt thereof.
Particular preference is also given to a compound of formula I wherein
T is NH or especially O;  means a radical selected from the radicals of formulae 
Z is oxygen orxe2x80x94Zxe2x80x94 is a bond, and Y and Y* or xe2x80x94Y and xe2x80x94Y* are defined as hereinbefore or hereinafter, especially either Y is hydroxy and Y* is hydrogen, or xe2x80x94Y and xe2x80x94Y* together form a bond, or a salt thereof.
Especially preferred are the compounds named in the examples, or salts thereof (especially pharmaceutically acceptable salts), provided that a salt-forming group is present.
The compounds of formula I may be prepared by methods known per se preferably in that
a) an acid of formula II, 
wherein T* is hydroxy, NH2, NH(alkyl) or N3 and (alkyl), A, Z and R have the meanings given for compounds of formula I, and wherein functional groups that should not participate in the reaction (especially the two OH groups at C-3 and C-6) are present if necessary in protected form, is cyclised, if T*xe2x95x90N3, this taking place after reduction of the azide group, and then if necessary any protecting groups are removed,
and, if desired, an obtainable compound of formula I is converted into a different compound of formula I; an obtainable free compound of formula I is converted into a salt; an obtainable salt of a compound of formula I is converted into another salt or the free compound of formula I; and/or obtainable isomeric mixtures of compounds of formula I are separated into the individual isomers;
In the following description of the detailed process conditions, the starting products and the reactions, if not otherwise stated, T, A, X, Y, Z, R, Rk and Rk* have the meanings given for compounds of formula I.
Process a):
A compound of formula II may be present in free form, or if the reaction of functional groups which should not participate in the reaction is to be prevented, in a form in which the functional groups that do not participate are present in protected form.
If one or more other functional groups, for example hydroxy or amino, in a compound of formula II are or need to be protected, because they should not take part in the reaction, these are those usually used in the synthesis of peptide compounds, and also of cephalosporins and penicillins, as well as nucleic acid derivatives and sugars. The protecting groups may already be present in precursors and should protect the functional groups concerned against unwanted secondary reactions, such as acylations, etherifications, esterifications, oxidations, solvolysis, and similar reactions. The protecting groups for functional groups in starting materials whose transformation should be avoided, in particular hydroxy or amino groups, include especially the conventional protecting groups that are normally used in the synthesis of peptide compounds, cephalosporins, penicillins or nucleic acid derivatives and sugars. In certain cases, the protecting groups may, in addition to this protection, effect a selective, for example stereoselective, course of reactions. It is a characteristic of protecting groups that they lend themselves readily, i.e. without undesired secondary reactions, to removal, typically by solvolysis, reduction, photolysis or also by enzyme activity, for example under conditions analogous to physiological conditions, and that they are not present in the end products. The specialist knows, or can easily establish, which protecting groups are suitable for the reactions mentioned hereinabove and hereinafter.
The protection of functional groups by such protecting groups, the protecting groups themselves, and their removal reactions are described for example in standard reference works, such as J. F. W. McOmie, xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, Plenum Press, London and New York 1991, in T. W. Greene, P. G. M. Wuts, xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, 2nd edition, John Wiley and Son Inc., 1981, in xe2x80x9cThe Peptidesxe2x80x9d; Volume 3 (E. Gross and J. Meienhofer), Academic Press, London and New York 1981, in xe2x80x9cMethoden der organischen Chemiexe2x80x9d (Methods of organic chemistry), Houben Weyl, 4th edition, Volume 15/I, Georg Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, xe2x80x9cAminosxc3xa4uren, Peptide, Proteinexe2x80x9d (Amino acids, peptides, proteins), Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and in Jochen Lehmann, xe2x80x9cChemie der Kohlenhydrate: Monosaccharide und Derivatexe2x80x9d (Chemistry of carbohydrates: monosaccharides and derivatives), Georg Thieme Verlag, Stuttgart 1974.
Protecting groups are preferably used analogously to the groups named in the examples (especially hydroxy protecting groups of the tri-lower alkylsilyl type), and introduced analogously to the methods described therein, and removed if necessary.
Cyclisation may be effected under conventional conditions. If T* is a hydroxy group, then cyclisation corresponds to macrolactonisation. If T* is NH2 or NH(alkyl), then it corresponds to lactam formation. If T* is an azide group (N3), then first of all it must be reduced, and subsequently lactam formation is also effected. Alternatively, treatment of the azido acid with Ph3P may directly lead to the macrolactam.
The lactonisation (formation of a lactone of formula I or of a protected derivative thereof with Txe2x95x90O) from a compound of formula II (or from a protected derivative thereof) preferably takes place in the presence of a coupling medium, for example a compound that can convert the free acid of formula II into an activated form, for example by forming an anhydride or an acid halide, especially by a reaction with an acid halide such as an arylcarbonyl halide, especially arylcarbonyl chloride, whereby aryl means phenyl in particular, which is unsubstituted or is substituted once or many times, preferably up to three times, by a substituent preferably selected from halogen such as chlorine, nitro, lower alkoxy, lower alkoxy-carbonyl and cyano, and if necessary in the presence of a base, possibly a tertiary nitrogen base, such as a tri-lower alkylamine, e,g, triethylamine, and/or a di-lower alkylaminopyridine, such as N,N-dimethylaminopyridine, at preferred temperatures of between xe2x88x9210 and 100xc2x0 C., preferably between 0 and 75xc2x0 C. The reaction may also be carried out in such a way that first of all an activated form of the acid of formula II is produced, for example an anhydride, and then this anhydride is reacted to the corresponding lactone, whereby both reactions can also follow one another in one and the same reaction mixture. The reactions are preferably effected in suitable solvents or solvent mixtures, such as ethers, e.g. tetrahydrofuran, or aromatic hydrocarbons such as benzene or toluene.
The lactam formation (macro-lactamisation) is effected under conditions which are customary for linking carboxylic acid amide bonds, whereby coupling reagents conventional in peptide chemistry, such as DCC/HOBt, HBTU, TPTU, HATU inter alia may be employed. It may also take place using for example diphenylphosphoryl azide or bromotripyrrolidino-phosphonium hexafluorophosphate.
The reduction of the azide group (if T*xe2x95x90N3) takes place by methods known per se, in particular using triphenylphosphine or by catalytic hydrogenation (see also WO 99/02514, where known methods are described).
Reactions:
In the additional process steps, carried out as desired, functional groups of the starting compounds which should not take part in the reaction may be present in unprotected form or may be protected for example by one or more of the protecting groups mentioned hereinabove under process a). The protecting groups are then wholly or partly removed according to one of the methods described under process a).
The compounds of formula I, in which Z is O, may be produced from those compounds of formula I in which xe2x80x94Zxe2x80x94 is a bond which forms a double bond together with the adjacent bond, by means of epoxidation according to methods known per se, for example with a peroxide such as m-chloroperbenzoic acid or preferably dimethyl dioxirane H2O2 in the presence of catalytic amounts of CH3ReO3, or methyl-trifluoromethyl dioxirane, under conventional conditions, for example by reacting in a suitable solvent, such as a hydrocarbon, e.g. benzene, an ester such as ethyl acetate, a halogenated hydrocarbon, such as dichloromethane, a ketone such as acetone, a nitrile such as acetonitrile, water, or mixtures thereof, if desired in the presence of a complexing agent such as an ethylene diamine tetraacetate, e.g. disodium ethylene diamine tetraacetate, and/or a base such as a metal carbonate or metal hydrogen carbonate, e.g. sodium hydrogen carbonate, at a reduced temperature, for example in the range xe2x88x9280 to +10xc2x0 C., preferably from xe2x88x9250 to +5xc2x0 C.
Compounds of formula I in which Y is hydroxy and Y* is hydrogen may be converted, by eliminating water, into those compounds of formula I in which xe2x80x94Y and xe2x80x94Y* together form a bond that forms a double bond together with the bond present between the two binding carbon atoms. To do this, free OH groups (especially at C-3 and C-7) are preferably formylated with the assistance of the mixed anhydride of formic acid and acetic acid; subsequently, the formyl derivative is treated with DBU {1,8-diazabicyclo[2.2.2]undec-7-ene (1.5-5)} in dichloroethane, which leads to elimination of formic acid and the formation of a double bond between C-2 and C-3. Finally, the formyl protecting group is removed from the OH group at C-7 and from any further OH groups, for example with NH3/methanol.
Compounds of formula I, wherein T is O, may be converted into the corresponding com pounds of formula I in which T is NH or N(alkyl), by reacting the compound with Txe2x95x90O by forming a pi-allylpalladium complex, for example using palladium tetrakis triphenylphosphine, followed by treatment with a corresponding primary amine [NH3 or H2N(alkyl), or NaN3] and subsequent lactam formation as described under process a).
Salts of compounds of formula I with a salt-forming group may be prepared in a manner known per se. Acid addition salts of compounds of formula I may thus be obtained e.g. by treatment with an acid or with a suitable anion exchange reagent.
Salts can usually be converted to free compounds, e.g. by treating with suitable basic agents, for example with alkali metal carbonates, -hydrogencarbonates, or -hydroxides, typically potassium carbonate or sodium hydroxide.
Stereoisomeric mixtures, e.g. mixtures of diastereoisomers, can be separated into their corresponding isomers in a manner known per se by means of suitable separation methods. Diastereoisomeric mixtures may thus be separated into their individual diastereoisomers by means of fractionated crystallization, chromatography, solvent distribution, and similar procedures. This separation may take place either at the stage of one of the starting compounds or in a compound of formula I itself. Enantiomers may be separated through the formation of diastereoisomeric salts, for example by salt formation with an enantiomer-pure chiral acid, or by means of chromatography, for example by HPLC, using chromatographic substrates with chiral ligands. (Enantiomer separation is normally effected at the intermediate stage).
Starting Materials:
The starting materials are known, may be produced by known processes or are commercially available, or they may be produced as described in the following:
In the following preparation processes for intermediates, functional groups which are to be in protected form can be protected if necessary at suitable stages, whereby selective protection or deprotection is also possible. The protecting groups and the methods of introducing and/or removing them correspond to those named above under process a), especially those named in the above-mentioned standard reference works or, in particular, in the examples. As a rule, protecting groups are not mentioned in the following; the following examples show where the usage of the protecting groups is appropriate or necessary and can therefore be regarded as a preferred instruction as to when protecting groups should be used and if compounds should be produced with other radicals. In the following, protecting groups are not mentioned at all the points where they are appropriately used. The person skilled in the art is clear as to where this usage ought to or must occur.
For example, compounds of formula II, wherein T* is OH, are obtained whereby an 
wherein A, R and Z have the meanings given for compounds of formula I and PG is a protected hydroxy group, especially tri-lower alkylsilyloxy, such as tert-butyl dimethylsilyloxy, is reacted in the presence of a strong base, such as lithium diisopropylamide, in a suitable solvent such as an ether, e.g. tetrahydrofuran, at preferred temperatures of between xe2x88x9280 and 25xc2x0 C., preferably between xe2x88x9280 and 0xc2x0 C., with a carboxylic acid of formula IV 
wherein the OH group is in protected form (for example as ten-butyl dimethylsilyloxy ether) [if necessary, the protecting group is removed from the protected hydroxy groups either subsequently or later in the reaction sequence, as described above or analogously to the examples].
A compound of formula III may be produced, whereby an alcohol of formula V, 
wherein A, R and Z have the meanings given for compounds of formula I and PG has the meanings given for compounds of formula III, is oxidised under conditions that are generally known for the oxidation of primary alcohols to aldehydes, for example using oxalyl chloride, acetanhydride, trifluoroacetanhydride, dicyclohexylcarbodiimide, preferably oxalyl chloride and dimethyl sulphoxide, in a suitable solvent such as a halogenated hydrocarbon, e.g. dichloromethane, at a temperature of between xe2x88x92100 and 0xc2x0 C., preferably between xe2x88x9280 and xe2x88x9220xc2x0 C.
A compound of formula V may be obtained by reacting a compound of formula VI, 
wherein R, Z and A have the meanings given for compounds of formula I and the radicals PG, independently of one another, mean protected hydroxy, by means of removal of the protecting group from the primary OH group, for example as described above or analogously to the methods described in the examples.
A compound of formula VI may be obtained in that a compound of formula VII, 
(protected if required) wherein R and A have the meanings given for compounds of formula I and PG means protected hydroxy, is reacted with a compound of formula VIII, 
wherein PG means a protected hydroxy group. In this reaction, a Cu/Zn pair is preferably prepared in a suitable solvent such as an aromatic hydrocarbon, e.g. benzene, in the presence of ethylene bromide and a tri-lower alkylsilyl halide such as trimethylsilyl chloride, at an elevated temperature, e.g. between 30 and 90xc2x0 C., preferably at 90xc2x0 C.; after adding the compound of formula VIII in a suitable solvent, e.g. a dimethylacetamide/benzene mixture, and afterwards adding a tri-lower alkylsilyl triflate, such as trimethylsilyl triflate, and optionally further dimethylacetamide, at a temperature of between 30 and 90xc2x0 C., especially 60 to 70xc2x0 C., the reaction continues; then, tetrakis(triphenylphosphine)-palladium is added, and finally the compound of formula VII is added, if necessary with further solvent such as benzene, and the reaction is completed at preferred temperatures of between 30 and 90xc2x0 C., especially at 60xc2x0 C., whereby a compound of formula VI is obtained, in which xe2x80x94Zxe2x80x94 is a bond (if desired, this can be convened into the corresponding compound of formula VI in which Z is O under conditions analogous to those given below for the epoxidation reactions).
A compound of formula VII may be obtained in that an aldehyde of formula IX, 
(protected if required), wherein A and PG have the meanings given for compounds of formula VI, is reacted with a suspension of [Ph3PCHIR]+lxe2x88x92 (in which R has the meanings given for R in formula I), to which is added sodium hexamethyl disilazide (NaHMDS) which is dissolved in a suitable solvent such as a cyclic ether, e.g. tetrahydrofuran; the mixture is then stirred at temperatures of between xe2x88x9280 and xe2x88x9220xc2x0 C., and the compound of formula IX is added (preferably dissolved in the same solvent, e.g. tetrahydrofuran) and reacted in the same temperature range to form the compound of formula VII.
The aldehyde of formula IX is preferably obtained in that a compound of formula X 
(protected if required), wherein A has the meanings given for compounds of formula I and PG* is a hydroxy group (in which case an appropriate protecting group is added first of all, e.g. tri-lower alkylsilyl) or PG is protected hydroxy, is reacted with a hydride, especially with diisobutyl aluminium hydride, in a suitable solvent such as a halogenated hydrocarbon, especially dichloromethane, at preferred temperatures of between xe2x88x9220 and xe2x88x9290xc2x0 C., e.g. at xe2x88x9278 to xe2x88x9240xc2x0 C.
A compound of formula X may be preferably obtained in that an aldehyde compound of formula XI,
Axe2x80x94CHOxe2x80x83xe2x80x83(XI)
(protected if required),wherein A has the meanings given for compounds of formula I, is reacted with a reagent which is obtained, in particular, firstly by preparing a solution of a trilower alkylborane, such as triethylborane, in a suitable solvent such as a hydrocarbon, e.g. hexane, adding a sulphonic acid such as trifluoromethanesulphonic acid, then maintaining firstly temperatures of between 10 and 50xc2x0 C., and after adding a suitable halogenated hydrocarbon, such as dichloromethane, again maintaining the same temperature range, and finally mixing with (2R)-acetylbornane-10,2-sultam and preferably a base dissolved in the same solvent, especially a tri-lower alkylamine, such as Hxc3xcnig""s base; at a temperature of xe2x88x9280 to 0xc2x0 C., and then the aldehyde of formula XI is added, preferably dissolved in a suitable solvent such as a halogenated hydrocarbon, for example dichloromethane, and reacted in the latter temperature range.
Compounds of formula IV, formula VIII and aldehydes of formula XI are known, may be produced by processes known per se or are commercially available.
An alternative and preferred process for the production of a compound of formula II (especially a hydroxy-protected compound), wherein T* is OH and the remaining radicals have the meanings given for the compounds of formula I [in particular, wherein R means methyl, Prot means tert-butyldimethylsilyl, Y is hydroxy (or in protected form tert-butyl-dimethylsilyloxy) and Y* is hydrogen], starts with the following educts (with the respectively mentioned meanings, especially the preferred meanings):
By reacting a compound of formula XII, 
wherein Prot means a hydroxy protecting group, especially tert-butyldimethylsilyl, and the remaining radicals have the meanings given for compounds of formula I (protected if required), and removing the primary hydroxy protecting group, for example with tetrabutyl-ammonium fluoride in an ether such as tetrahydrofuran, at preferred temperatures of 0 to 50xc2x0 C., especially at room temperature, the corresponding protected compound of formula II is obtained.
The compound of formula XII is preferably obtained in that an ester of formula XII*, 
wherein the radicals have the above-mentioned meanings and E is alkyl, especially lower alkyl, such as methyl, or also aryl or aryl lower alkyl, is saponified, preferably with an alkali metal hydroxide, such as LiOH, in an alcohol such as isopropanol, in the presence of water and at temperatures of ca. 25 to 75xc2x0 C., especially 50xc2x0 C.
The compound Of formula XII* is preferably obtained by reacting an olefin of formula XIII 
wherein E has the meanings given for compounds of formula XII* and the remaining radicals have the meanings given for compounds of formula XII, with a halide of formula XIV, 
wherein Hal is halogen, especially iodine, Prot is a hydroxy protecting group and the remaining radicals have the meanings given for compounds of formula I, whereby preferably first of all the olefin is reacted with 9-borabicyclononane (9-BBN) in a suitable solvent, such as an ether, e.g. tetrahydrofuran, at temperatures of between 0 and 50xc2x0 C., especially at room temperature, and then the solution of the resulting trialkylborane is added to a mixture of a carbonate, especially cesium carbonate, in the presence of PdCl2(dppf)2 (dppf=diphenylphosphinylferrocen), triphenylarsine and XIV in a suitable solvent, such as dimethylformamide, and the reaction takes place at temperatures of between xe2x88x9225 and 30xc2x0 C., especially between xe2x88x9210xc2x0 C. and room temperature, to form the compound of formula XII*.
The compound of formula XIV is analogous to the compound of formula VII and may be produced in the same way (especially if Hal=l).
The compound of formula XII) is produced in that a compound of formula XV, 
wherein G is a protecting group, especially benzyl, and Y** is hydrogen and Yxe2x80x2 is protected hydroxy is firstly reacted with dicyclohexyl carbodiimide in the presence of dimethylaminopyridine and an alcohol Exe2x80x94OH, wherein E has the meanings given for compounds of formula XII* and means methyl in particular, if necessary, also in the presence of one or more further suitable solvents such as dichloromethane, at temperatures of between xe2x88x9230 and 40xc2x0 C., especially between xe2x88x9220 and 25xc2x0 C.; removal of the protecting group G from the product obtained, if G=benzyl this is effected with hydrogen (preferably at atmospheric pressure) in the presence of a catalyst, such as palladium on carbon, in a suitable solvent such as methanol or ethanol, preferably at temperatures of 0 to 50xc2x0 C., such as at room temperature, leads to the free alcohol; this is reacted with 2-NO2PhSeCN (Ph=phenyl) in the presence of tributylphosphine at 0 to 50xc2x0 C., preferably at room temperature, and is then treated with a base such as a carbonate, e.g. sodium hydrogen carbonate, and an oxidation agent such as hydrogen peroxide, at the same temperature.
The compound of formula XV may be preferably produced from a compound of formula XVI, 
wherein O-Prot has the meanings given for compounds of formula XII and the remaining radicals have the meanings given for compounds of formula XV, whereby first of all the protecting group Prot on the primary OH is removed, if Prot=tert-butyldimethylsilyl e.g. by treatment with camphorsulphonic acid in a suitable solvent, for example, a mixture of an alcohol such as methanol with a chlorinated hydrocarbon such as methylene chloride, at temperatures of between xe2x88x9210 and 25xc2x0 C., especially at 0xc2x0 C.; then the resulting alcohol is initially oxidised to the aldehyde, e.g. with (COCl)2 in dimethylsulphoxide in the presence of a base such as triethylamine, and in a chlorinated hydrocarbon such as methylene chloride, at a reduced temperature, preferably between xe2x88x9280 and xe2x88x9250xc2x0 C., especially at xe2x88x9278xc2x0 C.; then the aldehyde obtained is oxidised with NaClO2 in the presence of isobutene in a solvent mixture, such as tetrahydrofuran/tert-butanol/phosphate buffer pH 7 at temperatures of between 0 and 50xc2x0 C., especially at room temperature, to form the acid.
If a compound of formula XII* in which xe2x80x94Y and xe2x80x94Y* together form a bond is desired, this may be produced by eliminating the elements of water (if necessary after removing the protecting groups) from the obtained intermediate of formula XIII, in which Y is hydroxy (optionally protected) and Y* means hydrogen. In order to allow for elimination, in a compound of formula XIII wherein Y is protected hydroxy the protecting group has to be removed first.
A compound of formula XVI is preferably produced from a compound of formula XVII, 
wherein K means lower alkyl, especially methyl, or hydrogen, or the two groups K together form a 5- or six-membered alicyclic ring together with the binding carbon atom, and the remaining radicals have the meanings given for compounds of formula XVI, whereby first of all this compound is reacted with pyridinium-p-toluenesulphonate in a suitable solvent such as methanol, at temperatures of between 0 and 50xc2x0 C., especially at room temperature, and then the obtainable product is reacted by introducing the protecting group xe2x80x9cProtxe2x80x9d, for example with a trialkylsilyl trifluoromethane sulphonate, such as tert-butylsilyl-dimethylsilyl-O-trifluoromethane sulphonate.
The compound of formula XVII is produced in that a compound of formula XVIII, 
wherein G has the significance given for compounds of formula XV, but especially means benzyl, is added to the enolate of a compound of formula XIX 
which is formed at a temperature of preferably xe2x88x9280 to xe2x88x9250xc2x0 C., especially at xe2x88x9278xc2x0 C., by adding a strong base, such as LDA in a suitable solvent (LDA=lithium diisopropylamide), in which compound K is lower alkyl, especially methyl, or hydrogen, or the two groups K together form a 5- or six-membered alicyclic ring together with the binding carbon atom, and reacted with lithium diisopropylamide in a suitable solvent such as tetrahydrofuran, at the said temperatures, especially at xe2x88x9278xc2x0 C.
Compounds of formula XIX, especially those in which K means methyl, are known, see e.g. Chem. Eur. J. 2(11), 1477-1482 (1996).
A compound of formula XVIII is preferably produced by oxidation of an alcohol of formula XX, 
wherein G means a protecting group, such as benzyl.
Compounds of formula XX (for example with G=benzyl) are known or may be produced by known processes (see Synlett 1998, 861-864).
An alternative procedure for the preparation of a compound of the formula XIII is by reaction of a compound of the formula XXVII, 
with a compound of the formula XIX as defined above, which results in a compound of the formula XVII* 
wherein K and K is defined as described for compounds of the formula XIX. The compound of formula XVII* can then be converted to XIII by the same sequence of reactions as described for XVII except that removal of the protecting group G present in XVII and the following olefin-forming reaction are not necessary.
A compound of formula II, wherein T* is NH or N(alkyl), may be produced as follows:
Starting from a compound of formula X* 
which is obtained as a by-product in the preparation of the compound of formula X (see above), in which PG* is a hydroxy group and A has one of the meanings given for compounds of formula I, first of all the OH group is converted into a leaving group (e.g. tosylate, mesylate, inflate). This is then followed by a reaction with a source of azide, for example NaN3 or Bu3SnN3. A compound of formula XXI is obtained, 
wherein X* is the radical of formula 
xe2x80x83and A has the meanings given for compounds of formula I. This is converted into a protected amino compound by reduction followed by direct introduction of an amino protecting group or by alkylation and subsequent introduction of an amino protecting group to that a group NHxe2x80x94PR or N(alkyl)-PR is obtained, in which PR is an amino protecting group; or it is further handled in the form of the azide group.
The reduction of the azide to the amino group may take place e.g. with triphenylphosphine or by catalytic hydrogenation, and the introduction of the protecting group or alkyl group PR then follows.
A compound of formula XXII is obtained, 
wherein PR* is NHxe2x80x94PR or N(alkyl)-PR as defined above, or is N3 (identical to the compound of formula XXI), and A has the meanings given for compounds of formula I.
These are subsequently reacted to form the corresponding aldehyde of formula XXIII, 
wherein the radicals have the meanings given for compounds of formula XXII, e.g. with diisobutyl aluminium hydride.
An aldehyde of formula XXIII can also be obtained by initial reaction of (2S)-acetylbornane-1,2-sultam with an aldehyde A-CHO (XI) to provide the enantiomer of a compound of formula X (X**), which is then processed into XXIII in analogy to compound X*. Aldehyde XXIII is then converted to the corresponding vinyl halide with [RCHl-PPh3]+ lxe2x88x92 (analogously to the preparation of the analogous oxygen of formula VII, see above; R is as defined under formula I).
The iodide of formula XXIV thus obtained, 
wherein in the radicals have the meanings given for compounds of formula XXII, is then reacted with the above-described compound of formula XIII, wherein the radicals have the meanings given therein, whereby a compound of formula XXV is obtained, 
in which PR* has the meanings given for compounds of formula XXII and the remaining radicals have the meanings given for compounds of formula XII*. The reaction takes place analogously to that of the compound of formula XIII to form the compound of formula XII* (alkyl Suzuki coupling).
This is then followed by ester saponification to form a compound of formula XXVI, 
wherein the radicals have the meanings given for compounds of formula XXV. The reaction takes place under analogous conditions to those for the reaction of compounds of formula XII* to form those of formula XII.
Finally, macro-lactamisation follows. In this, coupling reagents that are customary in peptide chemistry may be used, for example DCC/HOBt, HBTU, TPTU, HATU inter alia [see also under process a)].
In this process,
(i) where PR* is a protected amino group, the protecting group must be already removed (in the case of tert-butoxycarbonyl e.g. with trifluoroacetic acid or HF/pyridine); alternatively, the N- and O-protecting groups may be removed simultaneously, or
(ii) where PR* is N3, the azide group must be reduced prior to macro-lactamisation, e.g. with triphenylphosphine or by catalytic hydrogenation.
In both cases, the corresponding compound of formula II is obtained, whereby according to (l), T*, NH or N(alkyl) is present, and according to (ii), NH is obtained.
Alternatively, the azidocarboxylic acid may be converted directly into the macrolactam through the action of triphenylphosphine at an elevated temperature.
Where present, hydroxy as Y in the above-mentioned compounds of formulae XII bis is preferably protected, for example as Prot-O, especially as tert-butyldimethylsilyloxy.
In compounds of formula II and their precursors, in which Z is present, xe2x80x94Zxe2x80x94 is preferably a bond (forms a double bond together with the adjacent bond), Compounds of formula I, in which Z is O, are then obtained preferably from the corresponding compounds of formula II, in which xe2x80x94Zxe2x80x94 is a bond.
In compounds of formula II, III, V, VI and/or VII, the bond indicated by a wavy line is understood to mean that the cis- or trans-form may exist, or a mixture thereof, the cis-form being preferred.
General Process Conditions
All process steps described here can be carried out under known reaction conditions, preferably under those specifically mentioned, in the absence of or usually in the presence of solvents or diluents, preferably those that are inert to the reagents used and able to dissolve them, in the absence or presence of catalysts, condensing agents or neutralising agents, for example ion exchangers, typically cation exchangers, for example in the H+ form, depending on the type of reaction and/or reactants at reduced, normal, or elevated temperature, for example in the range from xe2x88x92100xc2x0 C. to about 190xc2x0 C., preferably from about xe2x88x9280xc2x0 C. to about 150xc2x0 C., for example at xe2x88x9280 to 60xc2x0 C., at room temperature, at xe2x88x9220 to 40xc2x0 C. or at the boiling point of the solvent used, under atmospheric pressure or in a closed vessel, if required under pressure, and/or in an inert, for example an argon or nitrogen, atmosphere.
Salts may be present in all starting compounds and intermediates, if these contain salt-forming groups. Salts may also be present during the reaction of such compounds, provided that the reaction is not thereby disturbed.
At all reaction stages, isomeric mixtures that occur can be separated into their individual isomers, e.g. diastereoisomers or enantiomers, or into any mixtures of isomers,.e.g. racemates or diastereoisomeric mixtures, for example analogously to methods described under xe2x80x9cAdditional process stepsxe2x80x9d.
In certain cases, typically in dehydrogenation or aldol reactions, it is possible to achieve stereoselective reactions, allowing for example easier recovery of individual isomers.
The solvents from which those can be selected which are suitable for the reaction In question include for example water, esters, such as lower alkyl-lower alkanoate, e.g ethyl acetate, ethers, such as aliphatic ethers, e.g. diethylether, or cyclic ethers, e.g. tetrahydrofuran, liquid aromatic hydrocarbons, such as benzene or toluene, alcohols, such as methanol, ethanol or 1- or 2-propanol, nitrites, such as acetonitrile, halogenated hydrocarbons, such as methylene chloride, acid amides, such as dimethylformamide, bases, such as heterocyclic nitrogen bases, e.g. pyridine, carboxylic acids, such as lower alkanecarboxylic acids, e.g. acetic acid, carboxylic acid anhydrides, such as lower alkane acid anhydrides, e.g. acetic anhydride, cyclic, linear, or branched hydrocarbons, such as cyclohexane, hexane, or isopentane, or mixtures of these solvents, e.g. aqueous solutions, unless otherwise stated in the description of the process. Such solvent mixtures may also be used in working up, for example by chromatography or partitioning.
The invention relates also to those embodiments of the process in which one starts from a compound obtainable at any stage as an intermediate and carries out the missing steps, or breaks off the process at any stage, or forms a starting material under the reaction conditions, or uses said starting material in the form of a reactive derivative or salt, or produces a compound obtainable by means of the process according to the invention under the process conditions therein, and further processes the said compound in situ. In the preferred embodiment, one starts from those starting materials which lead to the compounds described hereinabove as preferred, particularly as especially preferred, primarily preferred, and/or preferred above all.
In particular, the invention also relates to intermediate products of formulae II, III, V, VI, VII, X and furthermore IX and XI, and also intermediate compounds of formulae XII*, XIII, XIV, XV, XVI and especially XXV and XXVI, wherein the radicals are respectively defined as mentioned, and especially have the preferred meanings for compounds of formula I; whereby instead of PG, if present, a free hydroxy group may also exist.
In the preferred embodiment, compounds of formula I are prepared analogously to the processes and process steps defined in the examples.
The compounds of formula I, including their salts, are also obtainable in the form of hydrates, or their crystals may include for example the solvent used for crystallisation (present as solvates).
Pharmaceutical Preparations, Methods, and Uses
The present invention relates also to pharmaceutical preparations that contain a compound of formula I as active ingredient and that can be used especially in the treatment of the diseases mentioned above. Preparations for enteral administration, such as nasal, buccal, rectal or, especially, oral administration, and for parenteral administration, such as intravenous, intramuscular or subcutaneous administration, to warm-blooded animals, especially humans, are especially preferred. The preparations contain the active ingredient alone or, preferably, together with a pharmaceutically acceptable carrier. The dosage of the active ingredient depends upon the disease to be treated and upon the species, its age, weight, and individual condition, the individual pharmacokinetic data, and the mode of administration.
The invention relates also to pharmaceutical preparations for use in a method for the prophylactic or especially therapeutic treatment of the human or animal body, to a process for the preparation thereof (especially in the form of compositions for the treatment of tumours) and to a method of treating the above-mentioned diseases, primarily neoplastic diseases, especially those mentioned above.
The invention relates also to processes and to the use of compounds of formula I for the preparation of pharmaceutical preparations which contain compounds of formula I as active component (active ingredient).
Preference is given to a pharmaceutical composition that is suitable for administration to a warm-blooded animal, especially a human or commercially useful mammal, suffering from a disease that is responsive to the inhibition of microtubule depolymerisation, for example psoriasis or especially a neoplastic disease, comprising a correspondingly effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof when salt-forming groups are present, together with at least one pharmaceutically acceptable carrier.
A pharmaceutical composition for the prophylactic or especially therapeutic treatment of neoplastic and other proliferative diseases of a warm-blooded animal, especially a human or a commercially useful mammal requiring such treatment, especially suffering from such a disease, comprising a new compound of formula I, or a pharmaceutically acceptable salt thereof, as active ingredient in a quantity that is prophylactically or especially therapeutically active against said diseases, is likewise preferred.
Pharmaceutical preparations contain from about 0.000001% to 95% of the active ingredient, whereby single-dose forms of administration preferably have from approximately 0.00001% to 90% and multiple-dose forms of administration preferably have from approximately 0.0001 to 0.5% in the case of preparations for parenteral administration or 1% to 20% active ingredient in the case of preparations for enteral administration. Unit dose forms are, for example, coated and uncoated tablets, ampoules, vials, suppositories or capsules. Further dosage forms are, for example, ointments, creams, pastes, foams, tinctures, lipsticks, drops, sprays, dispersions, etc. Examples are capsules containing from about 0.0002 g to about 1.0 g active ingredient.
The pharmaceutical preparations of the present invention are prepared in a manner known per se, for example by means of conventional mixing, granulating, coating, dissolving or lyophilising processes.
Preference is given to the use of solutions of the active ingredient, and also suspensions or dispersions, especially isotonic aqueous solutions, dispersions or suspensions which, for example in the case of lyophilised preparations which contain the active ingredient on its own or together with a carrier, for example mannitol, can be made up before use. The pharmaceutical preparations may be sterilised and/or may contain excipients, for example preservatives, stabilisers, wetting agents and/or emulsifiers, solubilisers, salts for regulating the osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dissolving or lyophilising processes. The said solutions or suspensions may contain viscosity-increasing agents, typically sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone, or gelatin, or also solubilisers, for example (copyright)Tween 80 [polyoxyethylene(20)sorbitan mono-oleate; trademark of ICI Americas, Inc, USA].
Suspensions in oil contain as the oil component the vegetable, synthetic, or semi-synthetic oils customary for injection purposes. In respect of such, special mention may be made of liquid fatty acid esters that contain as the acid component a long-chained fatty acid having from 8 to 22, especially from 12 to 22, carbon atoms, for example lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, arachidic acid, behenic acid or corresponding unsaturated acids, for example oleic acid, elaidic acid, erucic acid, brassidic acid or linoleic acid, if desired with the addition of antioxidants, for example vitamin E; xcex2-carotene or 3,5-di-tert-butyl-4-hydroxytoluene. The alcohol component of these fatty acid esters has a maximum of 6 carbon atoms and is a mono- or polyhydric, for example a mono-, di- or trihydric, alcohol, for example methanol, ethanol, propanol, butanol or pentanol or the isomers thereof, but especially glycol and glycerol. As fatty acid esters, therefore, the following are mentioned: ethyl oleate, isopropyl myristate, isopropyl palmitate, xe2x80x9cLabrafil M 2375xe2x80x9d (polyoxyethylene glycerol trioleate from Gattefossxc3xa9, Paris), xe2x80x9cLabrafil M 1944 CSxe2x80x9d (unsaturated polyglycolised glycerides prepared by alcoholysis of apricot seed oil and consisting of glycerides and polyethylene glycol ester; Gattefossxc3xa9, France), xe2x80x9cLabrasolxe2x80x9d (saturated polyglycolised glycerides prepared by alcoholysis of TCM and consisting of glycerides and polyethylene glycol ester; Gattefossxc3xa9, France), and/or xe2x80x9cMiglyol 812xe2x80x9d (triglyceride of saturated fatty acids of chain length C8 to C12 from Hxc3xcls AG, Germany), but especially vegetable oils such as olive oil, cottonseed oil, almond oil, castor oil, sesame oil, soybean oil and more especially groundnut oil.
The manufacture of injectable preparations is usually carried out under sterile conditions, as is the filling, for example, into ampoules or vials, and the sealing of the containers.
Pharmaceutical compositions for oral administration can be obtained, for example, by combining the active ingredient with one or more solid carriers, if need be granulating a resulting mixture, and processing the mixture or granules, if desired, to form tablets or tablet cores, If need be by the inclusion of additional excipients.
Suitable carriers are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations, and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and also binders, such as starches, for example corn, wheat, nice or potato starch, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, such as the above-mentioned starches, also carboxymethyl starch, crosslinked polyvinylpyrrolidone, alginic acid or a salt thereof, such as sodium alginate. Additional excipients are especially flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof.
Tablet cores may be provided with suitable, if need be enteric, coatings, using inter alia concentrated sugar solutions which may comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents or solvent mixtures, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of active ingredient.
Orally administrable pharmaceutical compositions also include hard capsules consisting of gelatin, and also soft, sealed capsules consisting of gelatin and a plasticiser, such as glycerol or sorbitol. The hard capsules may contain the active ingredient in the form of granules, for example in admixture with fillers, such as corn starch, binders, and/or glidants, such as talc or magnesium stearate, and if need be stabilisers. In soft capsules, the active ingredient is preferably dissolved or suspended in suitable liquid excipients, such as fatty oils, paraffin oil or liquid polyethylene glycols or fatty acid esters of ethylene or propylene glycol, to which stabilisers and detergents, for example of the polyoxyethylene sorbitan fatty acid ester type, may also be added.
Suitable rectally administrable pharmaceutical preparations are, for example, suppositories that consist of a combination of the active ingredient and a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alkanols.
The formulations suitable for parenteral administration are primarily aqueous solutions ([or example in physiological saline, obtainable by diluting solutions in polyethylene glycol, such as polyethylene glycol (PEG) 300 or PEG 400] of an active ingredient in water-soluble form, e.g. a water-soluble salt, or aqueous injectable suspensions containing viscosity-increasing agents, e.g. sodium carboxymethyl cellulose, sorbitol and/or dextran, and where appropriate stabilisers. The active ingredient, if need be together with excipients, can also be in the form of a lyophilisate and can be made into a solution before parenteral administration by the addition of suitable solvents.
Solutions such as those used, for example, for parenteral administration can also be employed as infusion solutions.
Preferred preservatives are, for example, antioxidants, such as ascorbic acid, or microbicides, such as sorbic acid or benzoic acid.
The invention similarly relates to a process or a method for the treatment of one of the above-mentioned pathological conditions, especially a disease which responds to an inhibition of microtubule depolymerisation, especially a corresponding neoplastic disease. A compound of formula I can be administered as such or in the form of pharmaceutical compositions, prophylactically or therapeutically, preferably in an amount effective against the said diseases, to a warm-blooded animal, for example a human, requiring such treatment, the compounds especially being used in the form of pharmaceutical compositions. In the case of an individual having a bodyweight of about 70 kg the dose administered is from approximately 0.1 mg to approximately 1 g, preferably from approximately 0.5 mg to approximately 200 mg, of a compound of the present invention. Administration is preferably effected e.g. every 1 to 4 weeks, for example weekly, every two weeks, every three weeks or every 4 weeks.
The present invention also relates in particular to the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, especially a compound of formula I named as a preferred compound, or a pharmaceutically acceptable salt thereof, as such or in the form of a pharmaceutical formulation containing at least one pharmaceutically employable carrier, for the therapeutical and also prophylactic treatment of one or more of the above diseases.
The present invention also relates in particular to the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, especially a compound of formula I named as a preferred compound, or a pharmaceutically acceptable salt thereof, for the preparation of a pharmaceutical formulation for the therapeutical and also prophylactic treatment of one or more of the above diseases.
The preferred dose quantity, composition, and preparation of pharmaceutical formulations (medicines) which are to be used in each case are described above.